The aim of this proposal is to study the regulation of gene expression in muscle development during embryogenesis in Drosophila melanogaster. Specifically, we will analyze two genomic recombinant DNA Drosophila muscle gene clones, lambda Dm 217 and lambda Dm 231, cloned in charon 4 lambda phage. One of these clones, Dm 217, encodes a Drosophila protein of 22,000 molecular weight. The mRNA encoding this protein is expressed in myoblast and myotube stages of embryogenesis. However, the mRNA for this protein is translated only during later myotube stages of embroyonic development. In addition one and possibly two other genes are encoded by this same DNA fragment. The mRNAs encoded by these genes are also expressed during early and late development. Clone Dm 231, on the other hand, encodes a protein of 25,000 molecular weight. the mRNA encoding this protein is expressed during early and late myogenesis. There are also two other linked genes on this clone and each is expressed only during early myogenesis and embryonic development. The structural characterization of these two clones will be determined by constructing restriction endonuclease maps for each by the techniques of single, partial, and double digests. The exact number and position of transcribed regions will be identified by Southern analysis. Coding regions will be subcloned in the pBR322 plasmid. The number of different transcripts, the size of mRNA, and the presence of large intervening sequences will be determined by R-looping. The expresion of these genes in myoblast, myotube, fibroblast, and primary muscle cell cultures treated with EGTA to inhibit fusion will be analyzed by Northern blots and kinetic hybridization. With these gene specific probes we will be able to rigorously analyze the specific steps in mRNA processing and evaluate potential splicing events involved in the regulation of expression of these genes. The kinectics of synthesis of these mRNAs will also be determined by pulse-labeling of mRNA in octane permeabilized embryos. The results of these studies will allow us to determine the relationship of gene linkage to the function of coordinately expressed genes, the contribution of transcriptional, post-transcriptional and translational control to their expression and the regulation of embryonic and muscle development.